Currently, diagnostic and screen procedures are used to detect and grade epithelial cancers and pre-cancers for the purposes of diagnosis and treatment. In the case of the cervix of the uterus the development of screening programs for cancer prevention often targets the early detection and identification of its curable precursors such as Cervical Intraepithelial Neoplasia (CIN).
The Pap-test is the primary screening method for cervical neoplasia. During this test, a large number of cells are obtained from the cervical epithelium, and are cytologically examined after appropriate fixation and staining. The accuracy of this method is limited by both sampling and reading errors, leading to a significant false negative rate. A great number of studies have been performed aiming to determine the performance of the Pap-test over the past years. Researchers agree that the mean sensitivity is 0.59 and the mean specificity is 0.69-0.75 [Nanda K et al. (2000) Annals of Internal Medicine, 16; 132(10): 810-819; Sankaranarayanana R, et al. (2005) International Journal of Gynecology and Obstetrics, 89:S4-S12; and Fahey M T et al. (1995) American Journal of Epidemiology, 141: 680-689]. It is also widely accepted that the Pap-test is unable to achieve concurrently high specificity and sensitivity. For example, a possible increase of specificity in the 0.90-0.95 range will result in a decrease of sensitivity in the 0.20-0.35 range [Fahey M T et al. (1995) American Journal of Epidemiology, 141: 680-689].
Typically, sensitivity (SS) and specificity (SP) are used as quantitative statistical parameters to describe the performance of diagnostic tests. The sensitivity expresses the percentage of the True Positives (TP), while specificity expresses the percentage of the True Negatives (TN). For example, a sensitivity of 80% (or 0.80) means that the test diagnoses correctly 80 of the 100 cases diagnosed as positive for the disease, with the aid of the gold standard test (the most definitive test or procedure against which other tests are measured).
In a routine clinical setting, an abnormal Pap stained smear is followed by colposcopy, which involves examination of the cervix using a low power microscope. The cervical tissue is evaluated according to the following criteria: a) the morphology of the lesion's margins; b) the vascular pattern of abnormal epithelium; and c) the degree of staining after topical application of a marker, such as an acetic acid solution. Colposcopic grading is based solidly on visual examination, and the detected lesions are classified according to empirically qualitative scales. Clinical diagnosis based on the visual assessment (colposcopy) features a sensitivity of 0.77 and a specificity of 0.64 [Mitchell M F, et al. (1998) Obstetrics & Gynecology, 91:626-631]. Conventional colposcopy fails to diagnose 56% of microinvasive and 30% of invasive cervical cancer, leading to an inability to treat the lesion at its curable state. In addition, there is a high level of disagreement among physicians in identifying sites with high grade neoplasias for biopsy. Researchers have reported a considerable inter-observer variability in identifying cervical lesions through colposcopy [Schiffman M, et al. (2003) Arch. Pathol. Lab. Med., 127: 946-949; NHS Report. Cervical Screening Programme, England: 2003-04 Statistical Bulletin 2004/20. October 2004. U.K.; and Cantor S B, et al. (1998) Obstetrics & Gynecology, 91;(2): 270-277]. This diminishes the reproducibility of colposcopy and it is mainly attributed to the fact that the colposcopic assessment is qualitative and subjective.
In order to obtain more accurate Cervical Intraepithelial Neoplasia (CIN) diagnosis and grading, biopsy samples are obtained from suspicious areas, which are then submitted for histological examination. Biopsy sampling poses several problems though, such as: a) subjectivity and high inter-observer disagreement (>30%), as revealed by the studies of Ismail et al. [Ismail S M, et al. (1989) British Medical Journal, 298;(6675): 707-710] Bellina et al. [Bellina J H, et al. (1982) South Med. J., 75;(1): 6-8. 56] and Robertson et al. [Robertson A J, et al. (1989) J. Clin. Pathol., 42;(3): 231-238], and b) risks of sampling errors in selecting an abnormal site for biopsy.
The existing diagnostic chain for cervical neoplasia has reduced the incidence and mortality to historically low levels but further substantial reduction seems unlikely with the existing diagnostic procedures. This fact highlights the need for alternative, more efficient technologies, implementing the stand alone, and single step “see and treat” concept.
Over the last decade there has been a considerable effort towards the development of novel optical technologies capable of providing improved and objective information for tissue pathology. These approaches are usually based on the fact that a tissue change from a normal to pathologic condition alters the tissue's structure and functionality, and these alterations can be detected in vivo, by exploiting the light-tissue interaction phenomena. The measurement and analysis of the characteristics of the remitted light from the tissue can also provide information about the presence of different molecules, or about the various structural and functional changes occurring during the progress of the disease, thus providing a means for the in vivo identification and grading of the lesion.
Previous attempts towards this direction include a variety of spectroscopic and spectral imaging techniques targeting the detection of biochemical and/or structural alterations in vivo. Indicatively, U.S. Pat. No. 4,930,516 discloses a method for detecting cancerous tissue, where a tissue sample is illuminated with excitation light at a first wavelength, producing a fluorescent radiation in response to the excitation light detected. The discrimination between cancerous tissue vs. normal tissue is based on the wavelength and amplitude of the emitted fluorescent radiation. Alternatively, the spectral amplitude of normal tissue will differ from that of a cancerous tissue at the same wavelength.
It is known that time resolved spectroscopy, which is based on monitoring the fluorescent decay time, has also a potential in discriminating the type, or condition, of an illuminated tissue. For example, U.S. Pat. No. 5,562,100 discloses a method for determining tissue characteristics based on illuminating a target tissue with a short pulse of excitation radiation at a particular wavelength, and detecting fluorescent radiation emitted by the target tissue in response to the excitation. Tissue characteristics are determined from the recorded amplitude of the emitted radiation. In a similar manner, U.S. Pat. No. 5,467,767 discloses a method for determining the malignant condition of a tissue, using time-resolved fluorescence spectroscopy.
Other methodologies focus on combining two or more measurement techniques to determine tissue characteristics. For instance, U.S. Pat. No. 6,975,899 discloses an apparatus and method utilizing fluorescence in combination with reflectance in order to de-couple the biochemical changes from the morphological changes occurring in a cancerous tissue. This combined approach is based on the fact that as tissue undergoes changes from a normal to a cancerous condition, fluorescence spectroscopy becomes less effective in determining tissue characteristics, as compared to absorption spectroscopy.
Other patents, such as U.S. Pat. No. 5,369,496, disclose a method and apparatus for diagnostic multispectral digital imaging using fluorescence, reflectance, and polarized reflectance spectroscopy. In U.S. Pat. No. 6,427,082 a method and a system is provided for discriminating healthy from pathologic cervical tissue based on the fluorescence response of the tissue to laser excitation (LIF), and the back-scattered response to illumination by white light.
In general, prior art spectroscopic methods focus on tissue characteristics at a limited number of points on the tissue, whereas optical imaging methods focus on time-independent measurements of optical parameters over the entire tissue area. Moreover, these methods provide information only for the altered biochemical or cellular tissue structure, and not for the altered functionality of the epithelium.
Another approach developed by C. Balas is substantially different than the conventional methodologies because it involves measuring quantitatively the dynamic phenomena occurring in tissues after the application of biomarkers (PCT Publication No. WO 01/72214 A1 [Balas C. (2001) IEEE Trans. on Biomedical Engineering, 48:96-104], and [Balas C J, et al. (1999) SPIE 3568: 31-37]). Measurements of the dynamic phenomena could potentially provide information for both structural and functional features of the tissue, facilitating an in vivo diagnosis.
Optical biomarkers are chemical substances that induce impermanent alterations of the optical response of the abnormal tissue. In the case of efficient biomarkers, the structural, morphological and functional alterations of the abnormal tissue are manifested in the optical signal generated during the biomarker tissue interaction facilitating lesion identification and localization.
A typical diagnostic procedure involving biomarker application includes administrating topically or systematically one or more biomarkers to tissue. Then, biomarker induced alterations in the optical properties of the tissue are visually observed qualitatively. Based on these alterations in the optical properties due to administration of the biomarker, abnormal areas are identified for diagnosis and treatment. Traditional diagnostic methods involving biomarkers suffer from several drawbacks mainly related to the fact that the visual assessment of dynamic optical phenomena cannot be effective, due to physiological limitations of the human optical system in quantitatively detecting and recording fast changing phenomena with different kinetics in different tissue sites.
The method and device disclosed in the aforementioned Balas reference relies on the administration of a pathology differentiating agent (biomarker), which has the property of enhancing the visualization of the altered structure and functionality of the abnormal cells selectively, and then the measurement at any spatial point and in various wavelength bands, the remitted light as a function of time. The recorded intensity of the remitted light (for example intensity of back-scattered light (IBSL), defuse reflectance (DR) and fluorescence intensity), as a function of time is defined as the ‘Dynamic Optical Curve’ (DOC), which expresses the temporal characteristics of the optical phenomena generated during the tissue-biomarker interaction. Modeling and analysis of the acquired DOC enables calculation of a variety of Dynamic Optical Parameters (DOPs) which characterize the biomarker-tissue interaction kinetics at every image location (pixel or group of pixels) which corresponds to a tissue location. The spatial distribution of these parameters comprises the kinetic map, which can be overlaid onto the color image of the tissue. These data could potentially provide a means for the in vivo detection, mapping and grading of the lesion for diagnosis, screening, and follow up, while simultaneously enabling guidance for biopsy sampling, and surgical treatment.
Typically, the clinical value of a diagnostic technique is partially determined by its performance both in terms of its sensitivity (SS) and specificity (SP) positive and negative predictive value. If the SS and SP are greater than those of the existing diagnostic methods, then a new method or procedure could be deemed suitable for screening and/or clinical diagnosis purposes.